This is a R21 proposal to investigate Phospholipase D4 (Pld4) a novel gene of unknown function with homology to classical PLD enzymes. Pld4 was chosen for study initially because its expression pattern appeared to be restricted to B cells and its mRNA levels changed in immature bone marrow B cells upon BCR stimulation. We now know that it is expressed most in certain myeloid lineage cells including plasmacytoid dendritic cells. We have recently generated a conditional knockout allele of Pld4. We find that Pld4 null mice have several subtle phenotypes, with provocative alterations in the immune system, including massive up-regulation of MHCII expression on a subset of macrophages, an increase in marginal zone B cell numbers, and a decrease in peritoneal B-1 cells. Antibody responses are subtly altered. The most striking functional phenotype we have identified is that Pld4 null mice are completely resistant to induction of experimental autoimmune encephalomyelitis (EAE). The exciting implication is that inhibition of the action of this enzyme may facilitate treatment of multiple sclerosis. Pld4 carries a conserved phospholipase D catalytic domain with active site histidine, lysine and aspartic acid (HKD motif), but otherwise differs significantly frm well-known family members such as PLD1 and 2. No catalytic activity has been established for Pld4. We propose a two-pronged approach to determine if the putative active site is important for biological function and to assess in which cell types Pld4 bioactivity is required for its multple effects. The long-term goal of this study is to understand the biology and biochemistry of this novel protein. PUBLIC HEALTH RELEVANCE: We recently generated mice carrying a mutation in the poorly characterized Pld4 gene and found that these mice are completely resistant to a mouse model of human multiple sclerosis. The implication is that elucidation of the function(s) of the Pld4 protein will lead the way to new treatments of multiple sclerosis by development of drugs that block its function. To approach this, we will determine in which cell types the protein must function to promote disease and test if missense mutations that are predicted to prevent Pld4 function as an enzyme catalyst are sufficient to prevent disease.